Thymic stromal cell specialization and the T-cell receptor repertoire

Ten years ago, we proposed a model for thymus function in which thymic epithelial cells are primarily responsible for imprinting major histocompatibility complex (MHC)-restricted specificity, and bone marrow-derived macrophages or dendritic cells are responsible for the induction of self-tolerance. Since then, transgenic and knockout models have allowed for a dissection of thymic stromal components in vivo, leading to a new understanding of their specialized functions. We have determined that with regard to class II-restricted CD4 T-cell development, two distinct subsets of thymic epithelium help shape the repertoire: Cortical epithelium appears solely responsible for positive selection, whereas a fucose-bearing subset of medullary epithelium is specialized for negative selection. This absolute separation of positive and negative selection into two distinct spatial and temporal compartments leads to a much simpler view of the process of repertoire selection. Finally, a novel view of the function of the thymic medulla is discussed.     

Antibiotic resistance in Staphylococcus aureus colonising the intestines of Swedish infants

Staphylococcus aureus has become a frequent coloniser of the intestinal tract of infants, but the health effects of such colonisation are not clear. In this study, the antibiotic resistance patterns of 116 S. aureus strains from the commensal intestinal microflora were determined. The strains were obtained from 81 Swedish infants who had been followed with regular stool samples and registration of antibiotic usage during their first year of life. The faecal population levels of the individual strains and the duration of their persistence in the microflora had been determined previously. The prevalence of antibiotic resistance among the 116 strains was modest: methicillin, 0%; penicillin G, 78%; erythromycin A, 3%; tetracycline, 2%; clindamycin, 0.9%; and fusidic acid, 0.9%. Colonisation by antibiotic-resistant strains was unrelated to antibiotic consumption by individual infants. Antibiotic-resistant strains were as capable of persisting in the intestinal microflora and reaching high faecal population levels as fully susceptible strains. No strain lost or acquired resistance during the colonisation period. Thus, antibiotic-resistant strains of S. aureus seem to be as fit for competition in the large bowel microflora as susceptible strains, even in the absence of selective pressure from antibiotics. This may aggravate the ecological consequences of antibiotic resistance development.     

Classification of Breast Masses Using Selected Shape,Edge-sharpness,and Texture Features with Linear and Kernel-based Classifiers

Breast masses due to benign disease and malignant tumors related to breast cancer differ in terms of shape, edge-sharpness, and texture characteristics. In this study, we evaluate a set of 22 features including 5 shape factors, 3 edge-sharpness measures, and 14 texture features computed from 111 regions in mammograms, with 46 regions related to malignant tumors and 65 to benign masses. Feature selection is performed by a genetic algorithm based on several criteria, such as alignment of the kernel with the target function, class separability, and normalized distance. Fisher's linear discriminant analysis, the support vector machine (SVM), and our strict two-surface proximal (S2SP) classifier, as well as their corresponding kernel-based nonlinear versions, are used in the classification task with the selected features. The nonlinear classification performance of kernel Fisher's discriminant analysis, SVM, and S2SP, with the Gaussian kernel, reached 0.95 in terms of the area under the receiver operating characteristics curve. The results indicate that improvement in classification accuracy may be gained by using selected combinations of shape, edge-sharpness, and texture features.     

Characterization of Thymic Nurse-Cell Lymphocytes,Using an Improved Procedure for Nurse-Cell Isolation

Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a maiority of immature CD4⁺8⁺3^low thymocytes, about 12% of apparently mature CD4⁺8^-3^high and CD4^-8⁺3^high thymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells.     

Selection for high and low mating propensity inDrosophila ananassae

InDrosophila ananassae, artificial selection was carried out for high and low mating propensity for 15 generations. Response to selection was from about F₅, with rapid divergence in mating frequencies in replicates of both fast and slow lines. To assess the effect of selection on the two sexes, females and males of the selected lines were tested against their respective counterparts of the control line after 15 generations. Significant differences in mating propensity were observed when selected males were tested against the control females, which suggests that males were much more affected by selection than females. After 15 generations the fast and slow lines (both replicates) were crossedinter se and mating frequencies of F₁ hybrids were studied in the same way as during the selection experiment. F₁ flies had a higher mating activity compared to their parental lines when males were derived from fast lines to produce hybrids. On the other hand, F₁ hybrids produced by crossing slow-line males with fast-line females showed mating frequencies similar to those of the slow parental lines. These findings suggest that mating propensity inD. ananassae is under the control of polygenes. Furthermore, the significant differences in mating propensity of hybrids produced by the fast and slow males indicate the possibility of a Y-linked influence on mating propensity inD. ananassae.The present investigation was carried out during the tenure of a senior research fellowship of the CSIR, New delhi, to S.C.     

Small CD4+8+TCRlow thymocytes contain precursors of mature T cells

To evaluate directly the developmental potential of cortical CD4⁺8⁺ thymocytes, highly purified populations of small, nondividing CD4⁺8⁺TCR^low and large, dividing CD4⁺8⁺TCR^high thymocytes from H-2^d mice expressing a transgenic T cell receptor restricted by H-2D^b (major histocompatibility complex class I) molecules were transferred into the thymus of normal, nonirradiated H-2^b recipient mice. The results show that both populations generate CD4^?8⁺ thymocytes under these conditions, thus providing conclusive evidence that small cortical thymocytes do not represent a “dead end” but an important intermediate stage in T cell development.     

Genetic variability of sexual behavior in a natural population ofDrosophila melanogaster

Although many experiments on laboratory stocks ofDrosophila have suggested that mate choice is a major feature of sexual selection in this organism, few attempts have been made to measure its extent in wild populations. In this study, a crossing design was used to obtain a set of 13 genetically identical independent lines representative of genotypes from an African population ofDrosophila melanogaster. They were tested for variation in sexual behavior using dyadic tests. Significant variation in orientation and vibration latencies was found for males, and in mating speed and copulation duration for both sexes. No evidence of assortative mating, either positive or negative, was found. The absence of a correlation in mating speed between males and females sharing the same genotype leads us to doubt the applicability of the notion of male eagerness and female reluctancy inDrosophila and the importance of vigor as a factor in mating speed. The absence of mate choice in natural populations ofDrosophila seems to us the most likely hypothesis on the basis of both theory and empirical evidence.     

Probe selection algorithm for oligonucleotide array-based medium-resolution genotyping

Medium-resolution genotyping has the goal of distinguishing different subgroups instead of each element in a group. An oligonucleotide array provides an inexpensive, high-throughput method to identify differences in DNA sequence among individuals, which is fundamental for genotyping. As the cost and difficulty of designing and fabricating the oligonucleotide array dramatically increase with the number of probes used, it is therefore important to have a design with a minimum number of probes meeting the requirement of medium-resolution genotyping. The first algorithm for designing and selecting probes for oligonucleotide array-based medium-resolution typing is reported. The goal in deriving the algorithm was to select a minimum number of probes from a large probe set on the premise of minimum loss of resolution. The algorithm, which was based on entropy, conditional entropy and mutual information theory, was used to select the minimum number of probes from a large probe set. The algorithm was tested on a human leukocyte antigen (HLA) sequence data set. Thirty probes were selected from 390 probes for HLA-A, and 60 probes were selected from 767 probes for HLA-B. Although the number of probes was reduced by almost ten times, the distinguishability was reduced only a little, by 0.45% (from 99.90% to 99.45%) for HLA-A and 0.27% (from 99.84% to 99.57%) for HLA-B, respectively. This is a satisfactory and practical result.     

Positive selection is an early event in thymocyte differentiation: high TCR expression by cycling immature thymocytes precedes final maturation by several days

T cell antigen receptor expression by cycling and post-cycling thymocytes has been analysed by flow cytometry. Normal mice were pulsed with 5-bromo-2'-deoxyuridine (BrdUrd), a thymidine analogue detectable with a monoclonal antibody. Thymocytes were surface-stained with antibodies against several V beta gene products and against whole alpha beta receptors and detection of BrdUrd in the nuclei was performed after enzymatic generation of single-stranded DNA. A significant (10%) percentage of thymocytes expressing high levels of alpha beta TCR were found in the cycle: these cells were immature, as shown by the CD4+8+ phenotype and by high HSA expression. After division, most alpha beta high BrdUrd+ cells entered a resting state and their number remained constant for 3 days, decreasing in two steps thereafter. This post-mitotic evolution was not modified by injection of an anti-mitotic drug. After day 4, a majority of the studied subset acquired a single positive phenotype. Location of BrdUrd+ V beta 8.2 high cells studied on frozen sections was found cortical at early times and medullary after day 3. V beta 6 expression by cycling and post-cycling thymocytes was analysed in various mouse strains, and early high expression by cycling thymocytes was found to be restricted to MIs 1b strains. These results suggest that high alpha beta TCR expression by cycling immature thymocytes corresponds to positive selection, which must therefore be considered as an early event in intrathymic differentiation.